ES2683032T3 - Methods of isolation, accumulation, characterization and / or identification of microorganisms using a sample filtration and transfer device, and said device - Google Patents

Abstract

An integrated sample filtration and transfer device comprising: an elongated and hollow body having a first end or tip that is provided with, or covered with, a filtration material, wherein said filtration material is located adjacent to and protrudes from said first end or tip; wherein said elongated and hollow body is filled or plugged with an adsorbent to provide support for the filtration material; and a second operable end to provide fluid flow for filtration.

Description

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DESCRIPTION

Methods of isolation, accumulation, characterization and / or identification of microorganisms using a sample filtration and transfer device, and said device

Field of the Invention

The present invention relates to methods and devices for isolating, accumulating, characterizing and / or identifying microorganisms in a test sample. In one aspect, the present invention relates to a method that makes use of a sample filtration and transfer device for isolation, accumulation, transfer and subsequent characterization and / or identification of microorganisms in a test sample. In another aspect, the present invention relates to devices for isolating, accumulating and / or purifying microorganisms from a sample.

Background of the invention

The detection of pathogenic microorganisms in biological fluids should be carried out in the shortest possible time, particularly in the case of sepsis, whose mortality remains high despite the wide range of antibiotics available to doctors. The presence of biologically active agents, such as a microorganism in a patient's body fluid, especially in the blood, is generally determined using blood culture bottles. Infections in the bloodstream are associated with high morbidity and mortality and, however, current diagnostic methods, cultures followed by biochemical identification and antibiotic susceptibility tests, may take several days to complete. In general, empirical therapy is initiated based on the clinical symptoms, and the results of the tests only influence the decisions of the physicians when the initial therapy fails. The ability to characterize infections in the bloodstream in the early hours, preferably not later than one hour after a positive blood culture result, will significantly increase the chemical importance of the diagnostic information provided. Molecular amplification methods have been proposed to meet this need, but serious difficulties remain with respect to this technique. The positive blood culture broth itself represents a naturally amplified population of microorganisms with potential for use in a variety of rapid identification (ID) tests.

Traditional automated phenotypic ID tests, such as Vitek®, Phoenix ™ and Microscan® systems, or manual phenotypic tests, such as APIs, require that microorganisms are in an adequate growth phase and free of product interference of the medium and blood in order to provide conclusive results. These systems employ colonies that have grown in positive broth for 18-24 hours in plating media. However, in an effort to obtain faster results, some laboratories have disclosed the use of these systems with microorganisms isolated from positive blood culture bottles. These direct tests from the bottle are not suitable for all microorganisms (eg, Gram-positive coconuts), are not validated by the test manufacturers, and generally take 3-8 hours to give results. Faster and more specific tests are urgently needed in order to provide the physician with the clinically relevant results within the first hours, preferably within the first hour after a positive culture result.

Mass spectrometry methods have the potential to allow the identification of microorganisms very quickly, but they can find interference from many compounds present in liquid microbiological culture media and in chemical samples, such as blood, or combinations thereof. The methods most commonly used to recover microorganisms directly from the positive blood culture broth are two-stage differential centrifugation and centrifugation in a serum separator tube.

Other methods have been described for the separation, characterization and / or identification of microorganisms, which include:

U.S. Pat. No. 6,177,266 discloses a method for the chemotaxonomic classification of bacteria with specific biomarkers of genus, species and strain generated by mass spectrometry analysis by flight time and matrix-assisted laser ionization / desorption source (MALDI-TOF-MS for its English version "matrix-assisted laser desorption / ionization time-of-flight mass spectrometry") with extracts of cellular protema or complete cells.

In US Pat. No. 7,070,739 presents a method to extract, separate and purify microbes that include viruses by two-dimensional ultra-centrifugation directly from body fluids or homogenized tissue. In a first stage of centrifugation, all the particles whose sedimentation rate is greater than those of the microbes to be identified are separated. In the second stage of ultracentrifugation, the isopnemic band is used in liquids that are introduced to form a wide range density gradient, using special serrated crank tubes. According to the patent, the separation technique can be used to detect bands in bands by dispersion of light or fluorescence using specific dyes of nucleic acids, and to recover the particles in bands in very small volumes for mass spectrometry characterization of protein subunits. viral and viral particles intact, and by determination

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Cytometric fluorescent flux of both the nucleic acid mass and the masses of fragments produced by restriction enzymes.

EP0533912A describes a sample pretreatment apparatus and a method for dialyzing fluid and urine. The patent application describes the use of a large-sized pore pre-filter to separate typical urinary sediments, such as blood cells, epithelial cells, cylinders, mucus and crystals. Any bacteria present passes through the pre-filter and is captured in a second down filter. Subsequently, the captured bacteria are accessed by manually disassembling the device.

U.S. Patent Application UU. Pub. No. 2007/0175278 describes the use of a liquid culture medium to grow a sample of interest, which includes, for example, blood, urine, feces, intravenous catheters, industrial production lines, water systems, a product food, a cosmetic product, a pharmaceutical product, and a forensic sample. Subsequently, the microorganisms can be collected from the liquid medium by methods known in the art, eg, by centrifugation. The concentrated microorganisms can then be transferred to the carrier material, optionally after drying, to obtain a vibrational spectrum. The patent application describes several methods for identifying and classifying microorganisms, which include vibrational spectroscopy, such as Raman spectroscopy.

However, these methods have several disadvantages when trying to separate and characterize microorganisms from some samples of chemical tests (eg, complex samples such as culture media containing blood). In the case of culture media containing blood, the resulting microbial preparations often contain red blood cells, platelets, lipid particles, plasma enzymes and contaminating cell debris, which can produce poor results. These methods are also very laborious and insecure due to stages that can result in aerosol exposure of potentially dangerous pathogens for the user.

U.S. Patent Application UU. Pub. No. 2010/0120085, of the same assignee as herein, describes methods to separate, characterize and / or identify microorganisms in a test sample. The method describes a sample preparation process comprising a selective lysis stage and a subsequent separation stage for the isolation and purification of an unknown microorganism from a test sample for the identification of the microorganism by mass spectrometry. The application also describes the use of filtration for the isolation and purification of an unknown microorganism from a test sample.

U.S. Pat. No. 5833927A discloses a device for capturing a component present in a fluid, wherein the device is a pipette tip having a rear end adapted to fit over a micro-pipette, and a front end with a membrane, adapted to bind to the component, which extends through the pipette tip.

U.S. Pat. No. 4999164 A discloses a pipetting device comprising a retention cone for holding a sliding pipette tip and a piston chamber, which communicates with a passage of the retention cone and contains a redproco piston, which acts on the tip of pipette that has been adjusted in the retention cone. A filter is provided in the device consisting of an aerosol retention filter that is removably assembled in a region comprising the lower end of the pipetting device, including the retention cone, as well as the highest end portion of the pipette tip under the retention cone. The filter can be designed in various ways and can have a progressive filter action. The filter may be disposed at the lower end of the retaining cone or at the pipette tip or in an interposed member.

Consequently, there remains a need for improved sample preparation methods, devices and / or kits for the isolation and / or accumulation of microorganisms from samples of chemical tests, which are compatible with rapid identification technologies such as the spectrometry of masses. In addition, the need for improved sample preparation methods, devices and / or equipment for simultaneous isolation and / or accumulation of a plurality of microorganisms from a plurality of samples of chemical tests, which are compatible with rapid and automated techniques, remains for the identification of microorganisms. The methods and devices described here produce a clean and concentrated sample of microorganisms that is optimal for analysis, for example, by mass spectrometry, especially for MALDI-TOF-MS analysis.

Compendium of the invention

The present invention provides methods and devices for isolation, separation and / or accumulation for the subsequent characterization and / or identification of microorganisms in a sample. The methods allow the characterization and / or identification of microorganisms faster than previous techniques, with the result of faster diagnoses (e.g., in a subject who has or is suspected of having sepsis, meningitis or a urinary tract infection) and faster identification of contaminated materials (eg, food or pharmaceuticals) or water. The steps involved in the methods of the invention, from obtaining a sample to the characterization and / or identification of microorganisms, can be carried out in a very time frame

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short to produce clinically relevant processable information, eg, in less than about 120 minutes.

In one aspect, the present invention relates to a method for isolating and subsequently identifying a microorganism in a test sample, comprising:

(a) provide a test sample that contains certainty or that may contain microorganisms;

(b) optionally lysing cells and / or non-microbial particulate material in said test sample producing a lysed sample;

(c) isolating said microorganisms from other components of said test sample or said lysate sample, and accumulating them, by filtration, using an integrated sample filtration and transfer device comprising:

a hollow and elongated body having a first end or tip that is provided with, or covered with, a filtration material, in which said filtration material is located adjacent and protrudes from said first end or tip, and in the that said elongated hollow body is filled or plugged with an adsorbent to provide support for the filtration material, and

a second operable end to provide fluid flow for filtration;

(d) transferring said sample of isolated microorganisms to a suitable container or slide to analyze and / or scrutinize said sample of isolated and accumulated microorganisms;

(e) analyzing said isolated or accumulated sample of said microorganisms to obtain measurements for the characterization and / or identification of said microorganism; Y

(f) characterize and / or identify said microorganisms in said isolated and accumulated sample based on the measurements obtained.

This document also describes a method to isolate and subsequently characterize and / or identify a microorganism in a blood culture, comprising:

(a) obtain a sample from a blood culture that contains certainty or that may contain microorganisms;

(b) optionally lysing cells and / or non-microbial particulate material in said sample to produce a lysed sample;

(c) isolating said microorganisms from other components of the lysed sample, and accumulating them, by filtration, using an integrated sample filtration and transfer device;

(d) transferring said sample of isolated microorganisms to a mass spectromethane plate;

(e) analyzing said isolated and accumulated sample of microorganisms by mass spectrometry to obtain a mass spectrum of said microorganisms; Y

(f) characterize and / or identify said microorganisms in said isolated and accumulated sample by comparing the acquired mass spectrum with reference mass spectra.

Also described herein is a method for isolating, and subsequently characterizing and / or identifying a microorganism in a urine sample, comprising:

(a) obtain a urine sample that contains certainty or that may contain microorganisms;

(b) optionally lysing cells and / or non-microbial particulate material in said urine sample to produce a lysed sample;

(c) isolating said microorganisms from other components of the optionally lysed sample, and accumulating them, by filtration, using an integrated sample filtration and transfer device;

(d) transferring said sample of isolated microorganisms to a suitable container or slide to analyze and / or scrutinize said isolated and accumulated sample of microorganisms;

(e) analyze said sample of microorganisms isolated or accumulated to obtain measurements for the characterization and / or identification of said microorganism; Y

(f) characterize and / or identify these microorganisms in the isolated and accumulated sample based on the acquired measurements.

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The isolated or accumulated sample of said microorganisms can be analyzed using spectroscopic screening, for example, based on intrinsic characteristics of the microorganisms (e.g. intrinsic fluorescence) or the vibrational structure of the constituent molecules (e.g., Raman spectroscopy) . In one embodiment of the invention, isolated or accumulated microorganisms can be analyzed by mass spectrometry (eg, MALDI-TOF MS).

In accordance with another embodiment of the invention, the integrated sample filtration and transfer device may comprise an elongated and hollow body (e.g., an elongated tube and cylindrical, hexagonal or similar configuration hollow) having a first end or tip which comprises or is provided with a filtration material (eg, a filtration membrane), wherein said filtration material is placed adjacent to and protrudes from said first end or tip, wherein said elongated hollow body is filled with, or plugged with an adsorbent to provide support to the filtration material, and a second end that works by providing fluid flow for filtration. For example, the second end may be intended for connection to a ford system, a syringe, a plunger or similar device to generate a ford. The hollow cylindrical body comprises a long and narrow body and can be made of glass, plastic, metal or other similar material. In yet another embodiment, as an alternative to the source of ford, the sample filtration and transfer device can use an internally plugged adsorbent to provide sufficient capillary action and / or suction force for filtration and washing. For example, the adsorbent can provide passive filtration to provide a capillary action or absorption force for fluid flow.

In another embodiment, the present invention also relates to an integrated sample filtration and transfer unit comprising a plurality of integrated sample filtration and transfer devices as described in the preceding paragraph with respect to isolation and / or accumulation of a plurality of test samples and for the simultaneous transfer of the plurality of isolated and / or accumulated microorganisms to a container, slides or plate to analyze said isolated or accumulated microorganisms.

Also described here is a two-part sample filtration and transfer system comprising a filtration unit that works for the isolation and / or accumulation of microorganisms for a plurality of test samples by filtration, and a transfer unit that works for the simultaneous transfer of said plurality of isolated and / or accumulated microorganisms to a slide or plate for analysis.

A kit for the isolation, accumulation and / or purification of microorganisms from a test sample comprising, in a concentrated combination, is also described:

(a) optionally a selective lysis buffer for the selective lysis of non-microorganisms known to be or may be present in a test sample;

(b) an integrated sample filtration and transfer device wherein said device functions for the isolation, accumulation and / or purification of microorganisms of a test sample, and for the subsequent transfer of microorganisms to a container, slide or plate for your analysis; Y

(c) at least one wash fluid or buffer to wash the sample of isolated, accumulated and / or purified microorganisms. The integrated sample filtration and transfer device may comprise an elongated and hollow body having a first end or tip that is provided with or covered with a filtration material, wherein said filtration material is adjacent and external to said first end or tip, and a second end to provide fluid flow for filtration.

The present invention is explained in more detail in the attached figures and the description below. Brief description of the drawings

Figure 1A shows a front view of an integrated sample transfer and filtration device, according to the present invention. Figure 2B shows a cross-sectional view of the integrated sample transfer and filtration device shown in Figure 1A.

Figure 2A shows a front view of a second design concept of an integrated sample filtration and transfer device, according to the present invention. Figure 2B shows a cross-sectional view of the device and Figure 2C shows a detailed view of a first end of the device.

Figure 3A shows an exploded view of the integrated sample transfer and filtration device illustrated in Figure 2 and Figure 3B shows a detailed view of a first end of the device.

Figure 4 shows a perspective view of a sample filtration and transfer unit for the isolation and / or accumulation of microorganisms in a plurality of test samples.

Figure 5 shows an exploded view of the sample filtration and transfer unit illustrated in Figure 4.

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Figure 6 shows an exploded view of a filtration unit, comprising the first part of a two part sample filtration and transfer system. This is not claimed as part of the present invention.

Figure 7 shows a perspective view of the filtration unit illustrated in Figure 6. This is not claimed as part of the present invention.

Figure 8 shows a partial enlarged view of the filtration unit illustrated in Figure 6. This is not claimed as part of the present invention.

Figures 9A-D show perspective views of a transfer unit. This is not claimed as part of the present invention. Figure 9A shows a perspective view of a slide or plate that is placed on the bottom seal plate. Figure 9B shows a perspective view of the bottom joint and the slide or plate. Figure 9C shows an enlarged view of the bottom joint aligned with the transfer pin block. Figure 9D shows a perspective view of the position of the lower joint plate at the top of the block of transfer pins and in alignment therewith.

Figure 10 shows a bottom view of the lower surface of the upper plate of the filtration unit illustrated in Figure 6. This is not claimed as part of the present invention.

Figure 11 shows an exploded view of the upper plate, gasket or intermediate belt and an upper block of the filtration unit illustrated in Figure 6. This is not claimed as part of the present invention.

Figure 12 shows an exploded and cross-sectional view of the upper plate, gasket or intermediate belt and an upper block of the filtration unit illustrated in Figure 6. This is not claimed as part of the present invention.

Figure 13 shows a schematic representation of a method comprising a lysis stage, a separation stage and transfer stage, facilitated with the integrated sample filtration and transfer device of Figure 1.

Detailed description of the invention

The present invention may comprise different embodiments and should not be construed as limited to the embodiments set forth herein in the description, but is only limited by the present claims. However, these embodiments in the description are presented here so that this disclosure is rigorous and complete, and to completely transfer the scope of the invention to those skilled in the art. For example, the features illustrated with respect to one embodiment may be incorporated into other embodiments, and the features illustrated with respect to a particular embodiment may be removed from that embodiment as long as they are contemplated within the scope of the present claims. In addition, numerous variants and additions to the suggested embodiments here will be apparent to those skilled in the art in light of the present description, which will not deviate from the present invention as defined in the appended claims.

Unless defined otherwise, all the technical and scientific terms used here have the same meaning as that normally understood by a person skilled in the art to which the invention belongs. The terminology used in the description of the invention is intended only to describe the particular embodiments and not limit the invention.

Definitions

As used here, "un," "una," or "el", "la" may mean one or more than one. For example, "one" cell can mean a single cell or a multiplicity of cells.

Also as used here, "and / or" refers to and includes any and all possible combinations of one or more of the associated items, as well as the absence of combinations when interpreted as an alternative ("or").

In addition, the term "approximately", as used herein when referring to a measurable value as an amount of a compound or agent of this invention, dose, time, temperature, and the like, will include variations of ± 20%, ± 10% , ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

As used herein, the term "microorganism" is intended to include organisms that are generally unicellular, which can be multiplied and manipulated in the laboratory, including, but not limited to, Gram-positive or Gram-negative bacteria, yeasts, molds, parasites, and mycoplasmas. Non-restrictive examples of Gram-negative bacteria of this invention include bacteria of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobaim, Stenotvophonas , Ralstonia, Achromobacter, Fusobacterium, Prevotella, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Brucella,

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Pasteurella, Providencia, and Legionella. Non-restrictive examples of Gram-positive bacteria of this invention include bacteria of the following genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Paenibacillus, Lactobacillus, Listeria, Peptostreptococcus, Propionibacterium, Clostridium, Bacteroides, Gardnecococcus Lectococcus, Kochinococcus, , Mycobacteria and Corynebacteria. Non-restrictive examples of yeasts and molds of this invention include those of the following genera: Candida, Cryptococcus, Nocardia, Penicillium, Alternaria, Rhodotorula, Aspergillus, Fusarium, Saccharomyces and Trichosporon. Non-restrictive examples of parasites of this invention include those of the following genera: Trypanosoma, Babesia, Leishmania, Plasmodium, Wucheria, Brugia, Onchocerca, and Naegleria. Non-restrictive examples of Mollicutes of this invention include those of the following genera: Mycoplasma and Ureaplasma.

In one embodiment, as described in more detail here, the microorganisms of a sample or growth medium can be "separated" or "isolated" and then scrutinized to characterize and / or identify the microorganism present in the sample. As used herein, the term "separate" is intended to encompass any sample of microorganisms that has been removed, concentrated or otherwise removed from their original state, or from a growth or culture medium. For example, according to this invention, microorganisms can be separated (eg, as a sample or mass separated from microorganism) from non-microorganisms or non-microbial components that may otherwise interfere with the characterization and / or identification. The term may include a layer of microorganisms collected on a solid surface (eg, a membrane of a filter). As such, a separate sample of microorganisms (or mass or thin film of microorganisms) may include any collection or layer of microorganisms and / or components thereof that are more concentrated, or otherwise more separate, than the original sample, and It can vary from a dense and very compact aggregate of microorganisms to a diffuse layer of microorganisms. Microbial components that may be comprised in a separate form or sample include, but are not limited to, pili, flagella, fimbriae, and capsules in any combination. Non-microbial components that are separated from microorganisms may include non-microbial cells (eg, blood cells and / or tissue cells), urinary cylinders or crystals and / or any component thereof. As used herein, the term "isolated" is intended to cover any sample of microorganisms that have been at least partially purified from their original state, or outside their growth or culture medium, and any non-microorganism or non-microbial component content in it. For example, according to this invention, microorganisms can be isolated (eg, as an isolated sample) from non-microorganisms or non-microbial components that may otherwise interfere with the characterization and / or identification. Non-microbial components that are separated from microorganisms may include non-microbial cells (eg, blood cells and / or tissue cells) and / or any component thereof.

In one embodiment, as described in more detail here, the microorganisms of a sample or growth medium can be "accumulated" or "captured" in or on a filter material (eg, a filter membrane), and then scrutinized for characterize and / or identify the microorganism present in the sample. As used herein, the term "accumulated" or "captured" comprises any sample of microorganisms that has been compressed or deposited in a mass or film of microorganisms. For example, microorganisms in a sample can be compressed or deposited in a mass or film on a filter material (eg, a filter membrane) by filtration. The term includes a collection of microorganisms (and / or components thereof) on the surface of a filter material (e.g., a filter membrane) after filtration (e.g., vacuum filtration). Components of microorganisms that may be comprised of compressed or deposited mass of microorganisms include, but are not limited to, pili, flagella, fimbriae, and capsules in any combination. According to this invention, microorganisms can be compressed or deposited in a mass (e.g., as a mass of substantially purified microorganisms), outside of non-microorganisms or non-microbial components that may otherwise interfere with the characterization and / or identification of the microorganism (e.g., by mass spectrometry). Non-microbial components that are affixed or separated from microorganisms may include non-microbial cells (eg, blood cells and / or tissue cells) and / or any component thereof.

As used herein, the expression "analyze said isolated or accumulated sample" is intended to include all known methods or means for analyzing, scrutinizing, obtaining or otherwise acquiring measurements or data that can be used for the characterization and / or identification of microorganisms ( for example, unknown microorganisms.) For example, an isolated or accumulated mass of microorganisms can be analyzed or examined by spectroscopic methods, eg, based on intrinsic characteristics of the microorganisms (e.g. intrinsic fluorescence) or vibrational structure of the constituent molecules (e.g., Raman spectroscopy) In another embodiment, an isolated or accumulated mass of microorganisms can be analyzed or examined by mass spectromethane methods (e.g., MALDI-TOF-MS) to obtain measurements or data that can be used for the characterization and / or identification of unknown microorganisms, as described in more detail here

The present invention provides methods for isolating or separating microorganisms, and subsequently characterizing and / or identifying microorganisms in a sample. Moreover, the method can be particularly useful for isolation or separation and the subsequent characterization and / or identification of microorganisms in complex samples, such as urine samples or culture media containing blood. Rapid methods also allow the characterization and / or identification of microorganisms more rapidly than prior techniques, and

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result in faster diagnoses (e.g., in a subject who has or is suspected of having sepsis, meningitis or a urinary tract infection) and the characterization / identification of contaminated materials (e.g., food and pharmaceuticals) or Water. The steps comprised by the methods of the invention, from the obtaining of a sample to the characterization / identification of the microorganisms, can be carried out in a very short period of time in order to obtain processically relevant processable information. In certain embodiments, the methods of the invention can be carried out in less than about 120 minutes, e.g., in less than about 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5 minutes The tremendous speed of the methods of the invention represents an improvement over the previous methods. The methods can be used to characterize and / or identify any microorganism like the one described here. In one embodiment, the microorganism is a bacterium. In another embodiment, the microorganism is a yeast. In yet another embodiment, the microorganism is a mold. In a further embodiment, the microorganism is a parasite. In another embodiment, the microorganism is a mycoplasma. In addition, the methods of the invention can be fully automated, thereby reducing the risk of handling infectious materials and / or contaminating the samples.

Samples

Samples that can be tested (ie, a test sample) by the methods of the invention include both chemical and non-chemical samples, where the presence and / or growth of microorganisms is suspected or suspected, as well as samples. of materials that are routinely or occasionally tested for the presence of microorganisms. The amount of sample used may vary widely due to the versatility and / or sensitivity of the method. The sample preparation can be carried out by any number of techniques known to the experts although one of the advantages of the present invention is that the types of complex samples, such as, for example, blood, body fluids and / or other substances opaque, they can be tested directly using the system with little or no thorough pretreatment. In one embodiment, the sample is taken from a culture. In another embodiment, the sample is taken from a microbiological culture (eg, a blood culture). In another embodiment, it is suspected or known that the sample contains microorganisms.

Chloric samples that can be analyzed include any type of sample generally analyzed in clinical or research laboratories, including, but not limited to, blood, serum, plasma, blood fractions, joint fluid, urine, semen, saliva, feces, fluid. cerebrospinal, gastric contents, vaginal secretions, vaginal secretions, tissue homogenates, bone marrow aspirates, bone homogenates, sputum, aspirates, swabs and rinses of swabs, other body fluids, and the like. In another embodiment, the chemical sample can be cultured, and the culture sample can be used.

The present invention is useful in research as well as in veterinary and medical applications. Suitable subjects from which clinical samples can be obtained are generally mairnferos subjects, but it can be any animal. The term "mamffer" as used herein includes, but is not limited to, humans, nonhuman primates, cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (eg, rats or mice) , etc. Human subjects include infants, children, youth, adults and geriatric subjects. Subjects from which samples can be obtained include, but are not limited to, mammals, birds, reptiles, amphibians and fish.

Non-chlorine samples that can be analyzed also include substances, which comprise, but are not limited to, food products, beverages, pharmaceuticals, cosmetics, water (e.g., drinking water, non-potable water, and wastewater), ballasts of seawater, air, soil, sewage, plant material (e.g., seeds, leaves, stems, roots, flowers, fruits), blood products (e.g., platelets, serum, plasma, fractions of white blood cells, etc. ), samples of donor organs or tissues, samples of bacteriological weapons, and the like. The method is also particularly well suited for real-time testing to monitor pollution levels, process control, quality control, and the like in industrial facilities. In another embodiment, the non-chemical sample can be cultured, and a culture sample can be used.

In one embodiment of the invention, samples are obtained from a subject (eg, a patient) who has or is suspected of having a microbial infection. In one embodiment, the subject has or is suspected of having septicemia, eg, bacteremia or fungemia. The sample may be a blood sample obtained directly from the subject. The sample can be obtained from a blood culture developed from a patient's blood sample, eg, a BacT / ALERT® blood culture. The blood culture sample can be obtained from a positive blood culture, eg, a blood culture indicating the presence of a microorganism. In certain embodiments, the sample is taken from a positive blood culture shortly after it appears as positive, e.g., within about 6 hours, e.g., within about 5, 4, 3, or 2 hours, or within about 60 minutes, e.g., about 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 minutes. In one embodiment, the sample is taken from a culture where the microorganisms are growing in the logantmic phase. In another embodiment, the sample is taken from a culture where the microorganisms are in a stationary phase.

In some embodiment, to assist in the recovery of adherent microorganisms, eg, from adsorbent particles, the well-known pretreatment steps can be employed for samples containing adsorbents. For example, a surfactant (e.g., Tween 80) can be added and the sample can be stirred in

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vortex. In other embodiments, the sample may also be sonicated to break the biofilms and release the intact microorganisms. Examples include S. aureus bound to particles of charcoal.

The sample volume must be large enough to produce an isolated and / or accumulated sample of microorganisms or a mass of microorganisms that can be analyzed and examined to obtain measurements for the characterization and / or identification of said microorganism after carrying out the separation / isolation stage of the methods of the invention. The appropriate volumes will depend on the source of the sample and the anticipated level of microorganisms in the sample. For example, a positive blood culture will contain a higher level of microorganisms by volume than a sample of drinking water to be examined for contamination, so that a smaller volume of blood culture medium may be necessary when compared to the water sample potable. In general, the sample size may be less than about 50 ml, eg, less than about 40, 30, 20, 15, 10, 5, 4, 3, or 2 ml. In certain embodiments, the sample size may be about 1 ml, eg, about 0.75, 0.5, or 0.25 ml. In certain embodiments where the separation is performed at the microscale, the sample size may be less than about 200 pl, eg, less than about 150, 100, 50, 25, 20, 15, 10, or 5 pl. In some embodiments (eg, when the sample is expected to comprise a small number of microorganisms), the sample size may be approximately 100 ml or more, eg, approximately 250,500, 750, or 1000 ml or more.

Optional lysis stage

In some embodiments, after obtaining a sample, the next step in the method of the present invention is to selectively lyse or dissolve the unwanted cells and / or material that may be present in the sample, e.g., blood cells and / or tissue cells. Cells and / or particulate material may be lysed or dissolved to allow separation and / or isolation of microorganisms from other components of the sample. The separation and / or isolation of microorganisms from other components prevents interference during the analysis or screening stage. If non-microbial cells are not expected to be present in the sample or are not expected to interfere with the examination stage, the lysis stage may not be necessary. In one embodiment, the cells to be lysed are non-microbial cells that are present in the sample. Typically, non-microbial cells that may be present in the sample are lysed. However, in some embodiments, selective lysis of specific classes of microorganisms may be desirable and, therefore, may be performed according to the methods described herein and that are well known in the art. For example, a class of unwanted microorganisms can be selectively lysed, e.g., yeasts are lysed while bacteria are not or vice versa. In another embodiment, the desired microorganisms are lysed in order to separate a particular subcellular component from the microorganisms, eg, cell membranes or organs. In one embodiment, all non-microbial cells are lysed. In other embodiments, a portion of the non-microbial cells are lysed, eg, cells sufficient to prevent interference with the screening stage. Cell lysis can be performed by any method known in the art to selectively lyse cells with or without Lysis of microorganisms, including, without limitation, the addition of a lysis solution, sonication, osmotic shock, chemical treatment, and / or a combination thereof.

A lysis solution is one capable of lysing cells, eg, non-microbial cells (eg, solubilizing or dissolving eukaryotic cell membranes) and / or microbial cells. In one embodiment, the lysis solution may comprise one or more detergents, optionally one or more enzymes, or a combination of one or more detergents and one or more enzymes, and may also include additional agents. In one embodiment, the detergent may be a non-denaturing liquid detergent, such as Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100 , Igepal® CA 630, Arlasolve ™ 200, Brij® 96/97, CHAPS, octyl-pD-glucopyranoside, saponin, and nonaethylene glycol monododecyl ether (C12E9, polidocenol). Optionally, denaturing organic detergents, such as sodium dodecyl sulfate, N-laurylsacosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetama-14, and C7BzO may be included. Optionally, solubilizers may be included, such as Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, non-detergent sulfobetamas (NDSB 201), amphiphols (PMAL-C8), and methyl-p-cyclodextrin. Typically, non-denaturing and solubilizing detergents are used at concentrations above their critical micellar concentration (CMC), while denaturing detergents can be added at concentrations below their CMC. For example, non-denaturing lactic detergents can be used at a concentration of about 0.010% to about 10%, e.g., from about 0.015% to about 1.0%, e.g., from about 0.05% to about 0, 5%, eg, from about 0.10% to about 0.30% (final concentration after dilution with the sample). In another embodiment, detergents comprising a "head" group of hydrophilic polyoxyethylene bonded to a "tail" group of hydrophobic alkane or alkene via an ether bond may be preferred. These detergents are commonly specified by the CxEy notation, where "x" is equal to the number of carbons in the alkane or alkene chain, while "y" is the number of oxyethylene monomers (CH2CH20) in the polyoxyethylene chain. Detergents of this type are preferred where x is in the range of 10-20 and e is in the range of 8-12. Detergents of this type are even more preferred where x is within the range of 12-18 and e is within the range of 9-11. For example, the alkane-polyoxyethylene or alkenopolyoxyethylene detergent may be selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, nonaethylene-glycol-monododecyl ether (polidocanol), or A combination thereof.

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Enzymes that can be used in lysis solutions include, without limitation, the enzymes that digest nucleic acids and other materials that clog the membranes (eg, proteinase, DNase, neuraminidase, polysaccharidase, Glucanex®, and Pectinex®). Other additives that may be employed include, without limitation, reducing agents such as 2-mercaptoethanol (2-Me) or dithiothreitol (DTT) and stabilizing agents such as magnesium, pyruvate, and humectants. The lysis solution can be buffered at any pH that is suitable to lyse the desired cells, and will depend on multiple factors, including, without limitation, the type of sample, the cells to be lysed, and the detergent employed. In some embodiments, the pH may be maintained in a range of from about 2 to about 13, e.g., from about 6 to about 13, e.g., from about 8 to about 13, e.g., from about 10 to about 13 Suitable pH buffers include any buffer capable of maintaining a pH in the desired range, eg, CAPS from about 0.05 M to about 1.0 M. For some types of samples (eg, urine), The optimum pH for the dissolution of cells and / or unwanted particulate material may be from about 2 to about 8.

In one embodiment, the sample and lysis solution are mixed and then incubated for a sufficient time for lysis and solubilization of cell membranes to occur, e.g., approximately 1.2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or 60 seconds, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 minutes or more, e.g., from about 1 second to about 20 minutes, from about 1 second to about 5 minutes, or from about 1 second to about 2 minutes. In another embodiment, the sample and lysis solution are incubated for approximately 30 seconds to approximately 5 minutes, or approximately 1 minute to approximately 3 minutes. The incubation time will depend on the strength of the lysis solution, e.g., the concentration of the detergent and / or the enzymes. In general, softer lysis buffers will require more time and greater dilution of the sample to completely solubilize non-microbial cells. The strength of the lysis solution can be selected based on the microorganisms known or suspected to be found in the mixture. For microorganisms that are more susceptible to lysis, a mild lysis solution can be used. Lysis can take place at a temperature of about 0 ° C to about 60 ° C, from about 15 ° C to about 40 ° C, from about 20 ° C to about 40 ° C, or from about 30 ° C to about 40 ° C.

In some embodiments, lysis conditions (eg, dissolution or incubation time), as well as separation and / or screening stages, may be sufficient to kill some or all microorganisms in the sample. The methods of the present invention are very versatile and do not require that all microorganisms be alive for isolation and identification to occur. In certain embodiments, some or all microorganisms may be dead, death occurring before, during and / or after the stages of the methods being carried out.

Other details and the description of lysis buffers contemplated in the practice of this invention are described in US Pat. Serial No. 12 / 589,929 (currently published as US 2010/0129857 A1), filed on October 30, 2009, with the title "Methods for Isolation and Identification of Microorganisms". Other details and the description of the lysis buffers contemplated can be found in US Pat. in process Serial No. 12 / 589,936 (currently published as US 2010/0120085 Al), filed on October 30, 2009, with the title "Methods for Separation, Characterization and / or Identification of Microorganisms using Mass Spectrometry".

Typically, in the practice of this invention, the lysis stage is carried out within a container (eg, a microcentrefuge tube). The container can be any container with sufficient volume to contain a test sample and optionally a lysis solution. The container can be a microfuge tube. In practice of this invention the separation device disclosed in the US patent application can be used. Serial No. 12 / 589,969 (currently published as Us 2010/0120133 A1), filed on October 30, 2009, with the title "Separation Device for Use in the Separation, Characterization and / or Identification of Microorganisms". The volume of the container may be from about 0.1 ml to about 25 ml, eg, about 1 ml to about 10 ml, eg, about 2 ml to about 8 ml. If the lysis stage and the subsequent isolation or separation stage are performed in microscale, the volume of the container may be from about 2 pl to about 100 pl, eg, about 5 pl to about 50 pl. The container may have a closure device attached or it may be threaded to accept a closure device (eg, a lid) so that the container can be tightly sealed during use. The presence of a closure reduces the risks of handling microorganisms that are or may be infectious and / or dangerous, as well as the risk of contaminating the sample. In addition, another possible advantage of the methods of the present invention is the ability to carry out one or more of the stages (e.g., lysis or filtration stages) with the microorganisms in a sealed container (e.g., a tightly sealed container). The present methods can avoid the health and safety risks associated with the manipulation of very virulent microorganisms, as occurs with the recovery of microorganisms from samples for direct testing.

Filtration and sample transfer devices

As described above, the present invention also relates to a sample filtration and transfer device that functions for the separation, capture and accumulation of microorganisms of a

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test sample by filtration to the ford, and the subsequent transfer of the captured and accumulated microorganisms (e.g., as a mass or a film) to a slide or test plate for the analysis or screening of the microorganisms (e.g., by mass spectrometna). In one embodiment, the sample filtration and transfer device comprises an integrated sample filtration and transfer device having an elongated and hollow body (e.g., an elongated tube and cylindrical, hexagonal or similarly shaped hollow), which has a first end or tip that is provided with or covered with a filtration material (eg, a filtration membrane), wherein said filtration material is adjacent to and protrudes from said first end or tip, wherein said body Elongated hole is filled with, or plugged with, an adsorbent to provide support for the filtration material, and a second end adapted for connection with a ford system or device. For example, the filtration material may be external with respect to the first end or tip of the elongated body (ie, it extends over or protrudes from it). The present applicants have found that the use of a filtration material that extends over or protrudes from the first end of the integrated sample filtration and transfer device allows the transfer of any of the isolated and / or accumulated microorganisms, e.g., by Rubbing or staining a plate or slide with the sample.

In one embodiment, the hollow and elongated body is made of glass. In another embodiment, the hollow and elongated body is made of a plastic material that is either rigid or semi-rigid, such as polypropylene (PP), polycarbonate (PC), polyethylene terephthalate (PET), or other plastic material. In general, the integrated sample filtration and transfer device comprises an elongated and generally cylindrical body whose diameter of the filtration tip is from about 0.5 mm to about 10 mm, from about 1 mm to about 5 mm, or from about 1.5 mm to approximately 3 mm. In another embodiment, the cylinder body can be widened to an even larger diameter to be able to contain a larger volume of filtered material. The filtration and transfer device can be 2 cm to about 20 cm in length, from about 3 cm to about 15 cm, or from about 4 cm to about 10 cm. In one embodiment, the integrated sample filtration and transfer device comprises an elongated cylindrical body having a diameter of about 1.5 mm to about 3 mm, and a length of about 4 cm to about 10 cm. In another embodiment, the integrated sample filtration and transfer device comprises an elongated cylindrical body having an internal volume of about 0.5 cm3 to about 10 cm3, about 1 cm3 to about 5 cm3, or about 1.5 cm3 to approximately 3.5 cm3. In another embodiment, the integrated sample filtration and transfer device comprises an elongated cylindrical body having a diameter of about 1.5 mm to about 3 mm and a length of about 4 cm to about 10 cm (or a volume of about 0 , 9 cm3 to approximately 4.7 cm3).

In one embodiment, the elongated, hollow cylindrical tube of the integrated sample filtration and transfer device can be filled or compacted with an adsorbent. The concentration of an adsorbent material behind the membrane is useful in two ways. First, it provides support, which allows the membrane to protrude slightly beyond the tip, the applicants having found that this allows a more efficient transfer of microorganisms from the filter material (e.g., a filter membrane) to a slide or target plate. Second, the use of an adsorbent in the filtration and transfer device also allows adsorption of the lysate (ie, media materials and / or culture cells) that has passed through the filtration material. Moreover, the applicants have discovered that the capping material provides a clear separation zone between the sample lysate and the filter membrane, which prevents back-mixing of the lysate filtrate in contact with the membrane during and after washing, thus preventing the recontamination of clean microbes. In general, any material can be used. For example, in one embodiment, the adsorbent can be a polyester, glass or cellulose material in the form of fibers or particles. In another embodiment, the adsorbent could be an adsorbent resin, a silica gel, a hydrogel, polyacrylic acid or polyacrylamide derivatives, vegetable gums, a molecular sieve, zeolite or other adsorbents well known to those skilled in the art.

According to the present invention, the first end or tip of the integrated sample transfer and filtration device is provided with, or covered with, a filtering material (or filtering material). For example, as described hereinabove, the filtration material (eg, a filter membrane) is adjacent to, and extends from, the first end or tip of the integrated sample transfer and filtration device. In general, any filter material with pore sizes that retain at least some portions of the microorganisms and allow the lysate to pass through them can be used in the practice of the invention. The filter materials used in the practice of this invention may comprise filter membranes or depth filters well known in the art. In one embodiment, the filter membrane will have a pore size of about 0.1 pm to about 30.0 pm, or about 0.1 pm to about 10.0 pm, or about 0.1 pm to about 1, 0 pm Examples of membranes may include, polyethersulfone (PES) membranes (eg, Supor® 200, Supor® 450, Supor® MachV (Pall-Gelman, Port Washington, NY), Millipore Express PLUS® (Millipore)). Other possible filter materials may include, HT Tuffryn® (polysulfone), GN Metricel® (mixed cellulose ester), Nylaflo® (Nylon), FP Verticel (PVDF), all of PallGelman (Port Washington, NY), and Nuclepore (polycarbonate ) by Whatman (Kent, UK). Examples of depth filter materials may include, type Gf / F, GF / C and GMF150 (fiberglass, Whatman), Metrigard® (fiberglass, PallGelman), AP15 (fiberglass, Millipore), as well as a variety of cellulose, polyester, polypropylene or other fiber filters or particulate filters, provided the filter media can retain a

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sufficient quantity of the target microorganisms to allow analysis. In another embodiment, a filter material formed of modified fibers or particles can be used, for example, a membrane with zeta potential.

As described above, the second end of the elongated hollow tube, opposite the first end or tip of the elongated hollow tube, can be fixed to a ford source or ford system, which functions to provide a ford for filtration (i.e., for filtration to the ford). In general, any means known in the art can be used to connect the sample filtration and transfer device to the ford system. For example, the filtration and transfer device can be connected to a ford system with the use of a simple ford tube, as is well known in the art.

In another embodiment, the sample filtration and transfer device may further comprise a compression bulb for manual application of a ford for filtration to the ford. The use of the compression bulb may also allow the use of a reverse flow technique to transfer a sufficient amount of microbes to a slide or plate for mass spectrometry analysis, as described here. In another embodiment, a syringe and a plunger can be used to generate a ford. In yet another embodiment, the integrated sample filtration and transfer device can use the internal adsorbent cap to provide sufficient capillary action and / or absorption force for filtration and washing (i.e., thus allowing passive filtration).

Referring now to Figure 1, an example of embodiment of the integrated sample filtration and transfer device is shown. Figure 1 illustrates an integrated sample filtration and transfer device 2 comprising a hollow and elongated cylindrical body or tube 4 having a first end or tip 6 and a second end 8. The first end or tip 6 is provided with, or capped with, a filter or filtration material 9, which functions to capture or accumulate microorganisms when vacuum or suction is applied to the integrated sample filtration and transfer device 2. In the present invention the filtration material 9 is adjacent and external to the ( that is, it extends over or protrudes from the) first end or tip 6 of the elongated cylindrical body or tube 4. The second end 8 is generally connected to a source or a ford system (not shown). In other embodiments, the second end 8 may be provided with a bulb or plunger to provide a suction force or fluid flow for filtration. As shown, in a possible embodiment, the cylindrical and hollow body can be filled or compacted with an adsorbent 10, as described above. According to this embodiment, the same adsorbent can provide a capillary action or absorbent force that provides the flow of fluids for filtration.

Another design concept is exemplified in Figures 2-3. Figures 2-3 illustrate an integrated sample filtration and transfer device 12 comprising an elongated and hollow cylindrical tube or body 14 having a first end 15 and a second end 16. The first end 15 is provided with, or capped with, a filter material 17, which functions to capture or accumulate microorganisms when vacuum or suction is applied to the integrated sample filtration and transfer device 12. In the invention, the filter material 17 is adjacent, and external to the first end or tip 15 of the elongated cylindrical tube or body 14 (ie, extends over or protrudes therefrom). The first end 15 of the integrated sample filtration and transfer device 12 may further comprise a tapered portion 18 and a flattened or flattened tip 19. In other embodiments, the second end 16 may be provided with a bulb or plunger to provide a force of suction for filtration. As also shown, in a possible embodiment, the cylindrical and hollow body can be filled or compacted with an adsorbent 10, as described above. According to this embodiment, the same adsorbent can provide a capillary action or absorbent force that provides the flow of fluids for filtration.

In another embodiment, the present invention provides a sample filtration and transfer unit comprising a plurality of integrated sample filtration and transfer devices for the isolation and / or accumulation of a plurality of test samples and for the simultaneous transfer of microorganisms isolated and / or accumulated in a container, slides or plate to analyze said isolated or accumulated sample of said microorganisms in order to obtain measurements for the characterization and / or identification of said microorganisms. This device is exemplified in Figures 4-5.

As shown in Figures 4-5, the sample filtration and transfer unit 20 comprises a base plate 22, an upper plate 24 and a pair of vertical support rods 26 located between the upper plate 24 and said base plate 22 and separating them. The upper plate 24 also comprises a pair of rollers 28 that allow the upper plate to move "up" and "down" in a vertical plane along the support rods 26.

As shown, the sample filtration and transfer unit 20 may further comprise a pair of base rails 30 and the corresponding rod rods 32 of the rack, which hold a rack 34 having a plurality of wells 36 to contain a plurality of tubes individual 38 (eg, microcentrefuge tubes). As shown, the base rails 30 may comprise grooves 40 that hold the rod rods 32 of the rack and allow the rod rods of the rack 34 to slide in a horizontal plane with respect to the base plate 22 and the base rails 28 As also shown, the rack 34 may comprise a crank 42 that allows the user or technician to slide the rack 34 back and forth in a horizontal plane, as guided by the slots 40, along the base rails 30 .

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In addition, as shown in Figures 4-5, the sample filtration and transfer unit 20 can also comprise a vertical axis arm 44 and a vertical platform 46, which allows the vertical axis support 44 to move "upwards" "And" down "(ie, in a vertical plane) together with vertical platform 46. This vertical movement allows the top plate to move" up "and" down "(ie, vertically) along with the support rods 26.

The top plate 24 supports a ford unit 50 that supports a plurality of integrated filter and sample transfer devices 52. As shown in Figures 4-5, the ford unit 50 may also comprise a horizontally oriented alignment bar 54 comprising a plurality of locations or wells 56 separated by equal spaces to contain a plurality of removable integrated filtration and sample transfer devices 52. As shown, in one embodiment, the alignment bar 54 contains or supports a plurality of ( eg, twelve (12)) integrated sample filtration and transfer devices 52. As shown, one or more separation points (eg, four (4)) 58 can be used to separate or support the alignment bar 54 from the top plate 24.

The ford unit 50 also includes a valve manifold 60 comprising a plurality of valves 62 and connectors 64 where each individually holds and connects an integrated sample filtration and transfer device 52 to the ford unit 50. Each individual valve 62 and connector 64 held on the valve manifold 60 is individually connected to a manifold 66 through individual ford tubes 68. The manifold 66 connects to a system to the ford (not shown) through the main pipe ford 70.

In operation, a ford is provided to each of the individual integrated filtration and sample transfer devices 52 from a ford source (not shown) through a ford channel. The ford channel comprises, in series from the ford source, the main ford tube 70 and the ford manifold 66. From the ford manifold 66, the ford channel connects to and supplies a ford to the feed channels individual ford, where each individual ford channel comprises, in series from the ford source, the individual ford tubes 68, valves 62, the valve manifold 60, the connectors 64, and finally each individual filter and transfer device integrated sample 52.

The present also describes, although without being part of the claimed invention, a two-part sample filtration and transfer system. The two part sample filtration and transfer system comprises a first part, or a filtration unit, for the isolation and / or accumulation of a plurality of test samples (i.e., test samples containing or suspected containing microorganisms) by filtration, and a second part, or transfer unit, for simultaneous transfer of the plurality of samples of microorganisms isolated and / or accumulated to a slide or a plate to obtain measurements for the characterization and / or identification of said microorganisms. This device is exemplified in Figures 69.

The two-part sample filtration and transfer system 100 may comprise a first part, or a filtration unit 102 (see, eg, Figure 6), and a second part, or transfer unit 104 (see, for eg, Figure 9D). As shown in Figures 6-12, the two-part sample filtration and transfer system 100 may comprise a 48-well filter unit 102 (Figure 6) and corresponding to the transfer unit 104 (Figure 9D) for transfer up to 48 filtered samples (i.e. masses of isolated and / or accumulated microorganisms) to a 48-well slide or plate (e.g., a 48-well MALDI-TOF plate) for analysis and subsequent characterization and / or Identification of up to 48 individual test samples (ie 48 individual masses of isolated and / or accumulated microorganisms). As one skilled in the art will appreciate, other well configurations are possible.

As shown in Figures 6-8, the filtration unit 102 comprises a ford base plate 106, a lower joint block 108 and an upper block 110. The base plate 106 comprises a ford connector 120 for attaching a conductor of ford (eg, a ford tube) to provide a ford to the two-part sample filtration and transfer system 100 from a ford source (not shown). The filtration unit 102 further comprises a plurality of removable bolts 112 and through holes of the bolts 114 that are provided through a ford base plate 106, a lower joint block 108 and an upper block 110. Bolts 112 and through holes in bolts 114 allow the filter unit 102 to be secured or assembled before or during the filtration. The filtration unit 102 also comprises a pair of alignment pins 116 and alignment through holes 118 that allow assembly of the filtration unit 102 before securing or assembling the unit 102 via bolts 112. As shown in Figure 6 , the through alignment holes 118, as well as the through holes of the bolts 114, are also provided through the ford base plate 106, a lower joint block 108 and an upper block 110.

The filtration unit 102 further comprises a ford gasket 122 that provides a tight seal between the ford base plate 106 and the lower gasket block 108.

As shown in Figure 6, the lower joint block 108 contains a filtration cavity 124 which comprises a space on the upper surface of the lower joint block 108 and therein contains a plurality of through filtration holes 140. The filtration cavity 124 houses a lower joint 128 and a higher joint

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130, which "sandwich" enclose a filter material 132 (eg, a filter membrane). As the cavity to the ford 124, the lowest and 128 highest joints 130 there comprise a plurality of through filtration holes 140, which correspond to the through filtration holes contained in the filtration cavity 124. As exemplified in Figure 6 , the filter cavity 124, the lower gasket 128 and the upper gasket 130 may comprise 48 corresponding through holes in blank 140 (again other sample orifice configurations are possible and are contemplated as part of the present invention). The lower 128 and upper 130 gaskets provide a tight seal that allows filtration through the filter material in response to the ford that is taken via the ford connector 120 and the ford source (not shown).

The upper block 110 may also comprise an intermediate joint or tape 136 and an upper plate 138, as shown for example in Figure 6. As the filter cavity 124, the lower gasket 128 and the upper gasket 130, the upper block 110 , the intermediate seal or tape 136 and an upper plate 138 comprise a plurality of holes or sample wells 142, in which a test sample can be added and filtered for isolation and / or accumulation of any of the microorganisms contained therein . Each between the plurality of holes or sample wells 142 corresponds to, or is aligned with, each of the through holes in ford 140 contained in the filter cavity 124, lower seal 128 and upper seal 130. Using a seal or tape intermediate 136, the upper block 110 and the upper plate 138 can be formed in two separate parts and then assembled with the tape or intermediate joint 136, which allows concave fluid flow channels to be formed (as shown in Figures 10- 12) in each part.

As shown in Figure 10, the bottom surface 180 of the top plate 138 may comprise a first fluid flow channel 182. The first fluid flow channel 182 illustrated in Figures 10 and 12 comprises an inlet 184 located in a side edge of the upper plate 138 and a plurality of distribution channels 186 that provide fluid communication between the inlet 184 and a plurality of individual sample holes or wells 142 in the upper plate 138. As more clearly shown in Figure 12, the first fluid flow channel

182 carries a flow of fluids, from inlet 184 to the upper edge of the holes or sample wells

individual 142 formed by the upper block 110, the seal or intermediate belt 136 and an upper plate 138. The first fluid flow channel 182 can be used to provide a liquid sample to the individual sample holes or wells, for example, the wells Individual sample 142 may be filled with a liquid solution or a wash buffer via the first fluid flow channel 182 (as shown, for example, by arrow 188).

As shown in Figure 11, the upper surface 190 of the upper block 110 may comprise a second fluid flow channel 192. The second fluid flow channel illustrated in Figures 11-12 comprises a

plurality of output channels 194 that carry from or connect the bottom of the plurality of holes or

individual sample wells 142 up to a plurality of distribution channels 196 contained in the upper surface 190 of the upper block 110, which in turn lead to an outlet door 198 contained in a side edge of the upper block 110. The second flow channel of fluids 192 can be used to withdraw fluid from individual sample wells 142 (as shown, for example, by arrow 200). For example, if one or more of the wells are blocked or clogged due to the accumulation of microorganisms in the filter material, the source of the ford can be disconnected, and a second means (e.g., a second ford) can be supplied to provide force or suction to extract excess fluid from individual sample wells 142 via the second fluid flow channel 192.

In operation, a plurality of test samples (e.g., lysed blood culture samples, according to a possible embodiment of the present invention) can be filtered through the filtration unit 102, and the microorganism contained therein can be isolated and / or accumulated in the filter material 132. According to this arrangement, an individual test sample (not shown) can be added to an individual sample well 142 and a ford can be applied to the filter unit 102 from a source of ford (not shown) via the ford connector 120 for the filtration of the lysed material through the filter material 132, whereby any of the microorganisms in the filter material 132 are loosened and / or accumulated. This procedure can be repeated with samples of different tests in each of the individual sample holes or wells 142 provided in the upper block 110.

As shown in Figures 9A-9D, the transfer unit 104 comprises a block of transfer pins 150 comprising a base 152, a pair of alignment pins 154 and a plurality of transfer pins 156. According to this arrangement, the Transfer pins 156 function to transfer a mass of isolated and / or accumulated microorganisms to a slide or plate 160 (e.g., a MALDI-TOF plate) by pressing the filter material (e.g., a filter membrane) firmly against the slide or plate 160, and thus transfer the isolated and / or accumulated microorganisms.

The transfer of isolated and / or accumulated microorganisms to a slide or plate 160 is illustrated in Figures 9A-9D. In operation, after filtration, the lower joint block 108 is removed from the filtration unit 102. The upper joint 130 is removed from the ford cavity 124 of the lower joint block 108 and a slide or plate 160 is placed ( eg, a MALDI-TOF plate) in the ford cavity on top of the filter material 132 (see Figures 9A and 9B). To provide the exact location of the slide or the plate 160 in the ford cavity 124, a rail 162 and a flange 164 are provided on the upper surface of the block of

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lower joint 108. Alignment of the slide or plate 160 with the gma 162 and the flange 164 ensures that the sample stains 166 contained on the surface of the slide or plate 160 are correctly aligned with the stains of corresponding microorganisms (not shown) that result from the filtration of individual test samples through the individual sample holes or wells 142 provided in the upper block 110 (i.e., each of the individual sample holes or wells 142 is aligned with a corresponding sample spot 166 ).

Next, the lower joint block 108, the filter material 132 and the slide or plate 160, are transferred to and aligned with the upper part of the transfer pin block 150 using the alignment pins 154 and the through alignment holes 118 provided in the lower joint block 108 (see Figure 9C). The alignment pins 154 and the through alignment holes 118 allow the transfer pins 156 to align correctly with the through holes 140 contained in the lower joint block 108, and thus allow proper alignment, in series, of the pins transfer, the microorganisms isolated and / or accumulated in the filter material 132 and the corresponding sample stains 166 contained on the surface of the slide or plate 160.

Finally, the transfer pins 156 can be pressed into the filter material 132 and the slide or plate 160, with the means known in the art, to transfer the isolated and / or accumulated microorganisms (result from the filtration of individual test samples to through one or more of the corresponding individual sample holes or wells 142) to the corresponding sample points 166 contained on the surface of the slide or plate 160 (see Figure 9D). Subsequently, the slide or plate 160 can be analyzed or examined to obtain measurements with respect to the characterization and / or identification of said microorganisms.

Separation, isolation and / or accumulation stage

The next stage in the method of the present invention (eg, the stage after lysing the sample, if a lysing or dissolution stage is performed) is a separation, isolation and / or accumulation stage. The separation, isolation and / or accumulation step can be carried out to separate and isolate or purify the microorganisms from other components of the sample (e.g., not microorganisms or components thereof) and to accumulate or capture the microorganisms. in a mass that can be transferred to a slide or mass spectromethane plate and subsequently examined for identification and / or characterization. The separation, isolation and / or accumulation do not have to be complete, that is, it is not required that 100% of the separation take place. It is only necessary that the separation, isolation and / or accumulation of the microorganisms from other components of the sample be sufficient to allow the analysis or examination of the microorganisms without substantial interference from the other components. For example, separation / isolation may result in a mass of accumulated or captured microorganisms that is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99 % pure or more.

In one embodiment, the separation, isolation and / or accumulation stage is performed as a filtration stage where a sample filtration and transfer device (as described herein) is placed in the sample (e.g., a lysed sample ) in a container and a vacuum is applied to the filtration and transfer device, which allows microorganisms to be separated, isolated and / or accumulated (e.g., microorganisms can be accumulated or captured in the filtration material (e.g. , a filter membrane) of the filtration and transfer device) from other components that may be present in the sample. According to this embodiment, other components of the sample (eg, no microorganisms or components thereof that may be present in the sample medium) pass through the filter or filtration material. Therefore, this filtration stage isolates, separates and / or accumulates the microorganisms from materials in the sample, such as media, cell debris, and / or other components that could interfere with the analysis or examination of the microorganisms (eg. , by mass spectrometna).

Accordingly, in one embodiment, a novel method is described for quickly processing microorganisms from a sample (e.g., a positive liquid culture), by means of a sample filtration and transfer device, for the characterization and / or identification of the microorganism. In one embodiment, the method comprises capturing and accumulating microorganisms on or in a filtering material and subsequently transferring the accumulated microbes to a target slide or plate for mass spectrometry analysis. With reference now to Figure 13, an exemplified method for separation / isolation, capture and accumulation, and the subsequent transfer of microorganisms for mass spectrometric analysis is shown. As shown in Figure 13, the method comprises the following steps: (1) obtaining a test sample that is known to contain, or may contain, a microorganism (eg, a positive blood culture) (labeled as step 1); (2) selectively lyse the non-microorganism cells in the test sample, and thus produce a lysed sample (step 2); (3) immerse a sample filtration and transfer device (as described herein) in a lysed sample (step 3); (4) apply a vacuum to the sample filtration and transfer device, so as to filter the lysed sample through the filter, and thus capture the microorganism in the filter material of the integrated filtration and transfer device (step 4); (5) transfer the filtration and transfer device to a washing fluid or buffer, to wash the filter (step 5); (6) wash the filter by applying or carrying a vacuum in the filtration and transfer device, where the washing fluid or buffer is taken through the filter, and, where it is washed so

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any of the microorganisms captured in the filter material (step 6); (7) transfer the filtration and transfer device to a MALDI-TOF target plate (described in more detail below) (step 7); (8) depositing microorganisms on the surface of the MALDI-TOF target plate (eg, using a small quantity application technique (dabbing)) (step 8); (9) add matrix solution to the sample of microorganisms on the plate (described in more detail below) (step 9); and (10) acquire a mass spectrum of the microorganism sample using MALDI-TOF (as described below) (not shown).

Optional transfer stage

After accumulating or capturing microorganisms on the filtration material (e.g., a filter membrane) of the filtration and transfer device, the next step, in an embodiment of the process, comprises the transfer and deposition of the accumulated microorganisms (i.e. , such as a mass or film) on a slide or target plate for analysis and / or examination (e.g., by mass spectrometry). According to the present invention, the sample filtration and transfer device, in addition to providing a device for capturing and accumulating microorganisms from a test sample, also serves as a transfer device, for transferring and applying microorganisms on a slide or a target plate for mass spectrometry analysis.

In one embodiment, the microorganisms accumulated on the filter and transfer device can be applied or directly deposited on a slide or a target plate. According to this embodiment, small amounts can be applied with the filtration and transfer device (e.g., vertically up and down) on a slide or a target plate, one or more times, to allow an amount to be transferred enough of microbes to the slide or plate for analysis. In some cases, the transfer of sufficient microbes for analysis may require the application of small amounts repeatedly.

In another embodiment, the microorganisms accumulated on the filtration and transfer device can be applied or directly deposited on a slide or a target plate using a reverse flow technique. The reverse flow technique comprises applying gentle reverse pressure through the integrated filtration and transfer device (e.g., with a compression bulb or a folded conduit) so that the tip exudes a small amount of liquid. The reverse pressure can be applied while small quantities are applied with the device vertically in or on the container, slide or target plate until enough liquid is released to leave approximately 1-2 pl of reverse flow suspension and thus transfer a sufficient amount of microbes for analysis by mass spectrometry.

In another embodiment, the microorganisms accumulated on the filter and transfer device can be applied or directly deposited on a slide or a target plate using an extension technique. The extension technique comprises extending or sliding the filtering material of the device through the surface of a slide or a target plate, one or more times, to allow a sufficient amount of microbes to be transferred to the slide or plate for spectrometry analysis. of masses In some cases, transfer of microbes sufficient for mass spectrometry analysis may require that the device be repeatedly extended or slid over the surface of the slide or target plate.

Analysis, measurement and / or exam stage

Once the microorganisms have been filtered (e.g., using the sample filtration and transfer device described here) for isolation and / or accumulation of the unknown microorganism, the mass of isolated and / or accumulated microorganism can be analyzed to obtain measurements for the characterization and / or identification of said microorganism. In one embodiment, the isolated or accumulated sample of said microorganisms can be analyzed using spectroscopic examination, e.g., based on the intnnsec characteristics of the microorganisms (e.g., intimate fluorescence) or the vibrational structure of the constituent molecules (e.g. ., Raman spectroscopy). In another embodiment, the isolated or accumulated microorganisms can be analyzed by mass spectrometry (eg, MALDI-TOF-MS). Other details and the description of the preferred methods for obtaining measurements for the characterization and / or identification of said microorganisms can be found in US Pat. in process herewith: (1) No. 12 / 589,952 (currently published as US 2010/0129858 Al), filed on October 30, 2009, with the title "Methods for Separation, Characterization and / or Identification of Microorganisms using Spectroscopy "; (2) No. 12 / 589,976 (currently published as US 2010/0156609 Al), filed on October 30, 2009, with the title "Methods for Separation, Characterization and / or Identification of Microorganisms using Raman Spectroscopy"; and (3) No. 12 / 589,936 (currently published as US 2010/0120085 Al), filed October 30, 2009, with the title "Methods for Separation, Characterization and / or Identification of Microorganisms using Mass Spectrometry".

The sample or mass of isolated and / or accumulated microorganisms can be examined spectroscopically. Optical spectroscopic methods can be used to analyze one or more intimate properties of the microorganisms, eg, a property present within the microorganism in the absence of additional agents, such as stains, inks, binding agents, etc. In other scenarios, optical spectroscopic methods can be used to analyze one or more foreign properties of microorganisms, e.g., a

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property that can only be detected with the help of additional agents. The examination can be carried out using, for example, fluorescent spectroscopy, diffuse reflectance spectroscopy, infrared spectroscopy, teraherz spectroscopy, transmission and absorbance spectroscopy, Raman spectroscopy, including Enhanced Raman Surface Spectroscopy (SERS) "Surface Enhanced Raman Spectroscopy"), spatially compensated Raman spectroscopy (SORS, for its English version "Spatially Offset Raman spectroscopy"), transmission Raman spectroscopy, and / or Raman resonance spectroscopy. To improve the Raman (SERS) and fluorescence signals, the microorganisms could be coated with gold and / or silver nanoparticles before centrifugation, and / or the internal optical surface could be pre-coated with metal colloids of size and shape individuals (Re: Lakowicz, Anal. Biochem. 337: 171 (2005) with respect to fluorescence; Efrima et al., J. Phys. Chem. B. (Letter) 102: 5947 (1998) with respect to SERS). In one embodiment, the sample or mass of isolated and / or accumulated microorganisms is analyzed to obtain useful measurements for the characterization and / or identification of the unknown microorganism, while the sample or mass remains in / on the filtration device and / or Sample transfer In another embodiment, as described herein, the sample or mass of microorganisms can be analyzed after transfer to a container, plate or slide.

In other embodiments, after filtering the sample (e.g., using a sample filtration and transfer device, as described herein), a portion of the sample is transferred onto a plate or slide for introduction into a mass spectrometer. . A highly absorbent substance is deposited on top of the sample (eg, matrix); This material has a very high optical absorption coefficient with respect to the laser frequency used to ionize the sample (e.g., for a nitrogen laser, the emission wavelength is 337 nm, so that the absorbent material it will have a high absorption coefficient at a wavelength of 337 nm). Once the sample and the absorbent substance have dried, the plate is inserted into the mass spectrometer. After the time necessary to reduce the sample (that is, to remove atmospheric gases from the sample so as to have an environment of 10-5 to 10-7 torr), the sample is introduced into the ionization chamber of the mass spectrometer . The sample is aligned with the system. When optimal alignment is achieved, nitrogen laser pulses are produced. The absorption of laser energy by the matrix causes it to separate from the surface of the plate due to the high energy deposited. As a side effect, the portions of the microorganism cells also vaporize and ionize in the process. These ions are accelerated to a known kinetic energy by generating an electrostatic field between the plate and the entrance to the spectrometer's flight tube (that is, this portion of the system is the mass / charge discriminator). All individually charged ions, regardless of mass, will have the same kinetic energy at the entrance to the flight tube, but will have speeds inversely proportional to their masses. From here, the ions go down the flight tube to the detector, and the lighter ions will arrive before the heavier ions (the flight tube is a mass / charge discriminator). The detector generates an electric charge every time an ion hits the detector. The detector's output is digitized and the output shows the mass / load ratio on one axis and the number of impacts on the other axis. In other embodiments, microorganisms transferred in the mass may be examined using mass spectrometry techniques, such as MALDI-TOF mass spectrometry, mass spectrometry with desorption ionization and electrospray ionization (DESI) for its English version "desorption electrospray ionization" ), mass spectrometry coupled to gas phase chromatography, mass spectrometry coupled to liquid phase chromatography, mass spectrometry with electrospray ionization (ESI), and spectrometry with a flow tube of selected ions (SIFT, for its English version “selected ion flow tube”).

In some embodiments of the invention, the characterization and / or identification of the microorganisms in the sample or isolated mass does not need to understand the identification of an exact species. The characterization includes the broad categorization or classification of biological particles as well as the real identification of a unique species. The classification of a microorganism from an isolated sample or mass may comprise the determination of the phenotypic, morphological and / or metabolic characteristics of the microorganism. For example, the characterization of biological parameters can be carried out based on observable differences, such as composition, form, size, aggregation and / or metabolism. In some embodiments, the classification of the biological particles of interest may not require prior knowledge of the characteristics of a given biological parameter, but only correlations compatible with empirical measurements, which makes this method more general and easily adaptable than the methods. based on specific binding events or metabolic reactions. As used here, "identifying" means determining to which family, genus, species and / or strain a previously unknown microorganism belongs. For example, identify a previously unknown microorganism at the family, gender, species, and / or strain level.

In some cases, the characterization includes classification models that provide useful information regarding the action to be taken. Additional details and description of the classification models contemplated can be found in US Pat. also in process No. 12 / 589,936 (currently published as US 2010/0120085 Al), filed on October 30, 2009, with the "Methods for Separation, Characterization and / or Identification of Microorganisms using Mass Spectrometry". alff, the preferred classification models comprise clusters in one or more of the following: (1) Gram Groups; (2) Chemical Gram Groups; (3) Therapeutic Groups; and (4) Functional Groups.

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Team.

The present application also describes, although not claimed, an equipment for isolation, accumulation and / or purification of microorganisms from a test sample. In its simplest form, the equipment will include: (1) optionally a non-microorganism lysis solution or lysis known to be present or may be present in a test sample; (2) an integrated sample filtration and transfer device (as described herein) for isolation, accumulation and / or purification of microorganisms that may be in a test sample, and for subsequent collection and transfer of microorganisms; and (3) at least one wash fluid or buffer to wash the isolated, accumulated and / or purified microorganism sample. The integrated sample filtration and transfer device used in the equipment is described in detail here.

Optionally, the equipment may include a selective lysis solution or buffer to selectively lyse or dissolve cells and / or unwanted particulate material that may be present in the test sample, eg, blood cells and / or tissue cells. Cells and / or particulate materials can be lysed or dissolved to allow separation and / or isolation of microorganisms from other components of the sample. The separation and / or isolation of microorganisms from other components prevents interference during any subsequent direct test application, for example, the analysis or examination with respect to the characterization and / or identification of the microorganism (e.g., by spectrometry). of masses) or the realization of microbial PCR of wide range on broth of blood culture. However, if it is not expected to find non-microorganism cells present in the sample or they are not expected to interfere with any subsequent assay, the lysis step may not be necessary.

In general, the selective lysis solution can be used to lyse or dissolve non-microorganism cells that may be present in the test sample. However, in other embodiments, selective lysis of specific classes of microorganisms may be convenient and therefore carried out in accordance with the methods described herein and as known in the art. For example, a class of unwanted microorganisms can be selectively lysed, e.g., the yeast is lysed while the bacteria are not or vice versa. In another embodiment, the desired microorganisms are lysed in order to separate a particular subcellular component from microorganisms, eg, cell membranes or organs. In one scenario, all non-microbial cells are lysed. In other scenarios, a portion of the non-microbial cells are lysed, eg, enough cells to prevent interference with the examination stage. The lysis of the cells can be carried out by any method known in the art as effective for selectively lysing cells with or without lysis of microorganisms, including, without limitation, the addition of a lysis solution, sonication, osmotic shock, chemical treatment, and / or a combination of the above.

The lysis solution is one capable of lysing cells, eg, non-microorganism cells (eg, by solubilizing or dissolving eukaryotic cell membranes) and / or microorganism cells. In one embodiment, the lysis solution may comprise one or more detergents, optionally one or more enzymes, or a combination of one or more detergents and one or more enzymes, and may also include additional agents. In one embodiment, the detergent may be a non-denaturing liquid detergent, such as Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100 , Igepal® CA 630, Arlasolve ™ 200, Brij® 96/97, CHAPS, octyl-UD-glucopyranoside, saponin and nonaethylene glycol monododecyl ether (C12E9, polidocanol). Optionally, denaturing organic detergents, such as sodium dodecyl sulfate, N-laurylsarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetama-14 and C7BzO, may be included. Optionally, solubilizing agents can also be included, such as Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, non-detergent sulfobetamas (NdSb 201), amphiphols (PMAL- C8) and methyl-U-cyclodextrin. Typically, non-denaturing and solubilizing detergents are used in concentrations above their critical micellar concentration (CMC), while denaturing detergents can be added at concentrations below their CMC. For example, denaturing plastic detergents can be used at a concentration of about 0.010% to about 10%, e.g., from about 0.015% to about 1.0%, e.g., from about 0.05% to about 0, 5%, eg, from about 0.10% to about 0.30% (final concentration after dilution with the sample). In another scenario, detergents comprising a "head" group of hydrophilic polyoxyethylene bonded to a "tail" group of hydrophobic alkane or alkene via an ether bond may be preferred. These detergents are commonly specified using the indication of the CxEy form, where "x" is equal to the number of carbons in the alkane or alkene chain, while "y" is the number of oxyethylene monomers (CH2CH2O) in the polyoxyethylene chain . Detergents of this type are preferred where x is in the range of 10-20 and e is in the range of 8-12. Even more preferred are detergents of this type where x is within the range of 12-18 and e is within the range 9-11. For example, the alkane-polyoxyethylene or alkenopolyoxyethylene detergent may be selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, nonaethylene glycol monododecyl ether (polidocanol), or a combination thereof.

Enzymes that can be used in lysis solutions include, without limitation, enzymes that digest nucleic acids and other materials that clog the membranes (eg, proteinase, DNase, neuraminidase, polysaccharidase, Glucanex®, and Pectinex®). Other additives that may be employed include, without limitation, reducing agents such as 2-mercaptoethanol (2-Me) or dithiothreitol (dTt) and stabilizing agents such as magnesium, pyruvate and humectants. The lysis solution may be buffered at any pH that is suitable to lyse the cells.

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desired, and will depend on multiple factors, including, without limitation, the type of sample, the cells to be lysed, and the detergent used. In some embodiments, the pH may be from about 2 to about 13, e.g., about 6 to about 13, e.g., about 8 to about 13, e.g., about 10 to about 13. Buffers of suitable pH they include any buffer capable of maintaining a pH in the desired range, eg, about 0.05 M to about 1.0 M in CAPS. For some types of sample (eg, urine), the optimum pH for dissolving unwanted cells and / or particulate material may be from about 2 to about 8.

The kit will also include at least one wash fluid or buffer to wash the microorganism or mass of isolated, accumulated and / or purified microorganisms on the filter material. The wash buffer can also be used to separate, isolate or purify the accumulated or captured microorganisms, "eliminating" other components (eg, media, media component, cell debris, non-microorganisms or components thereof) that may be present. in the test sample As one skilled in the art will readily appreciate, the use of a wash buffer allows or facilitates the removal (or passage through the filter material) of medium, media components, cell debris, non-microorganisms or components thereof, which may otherwise interfere with subsequent tests (eg, mass spectrometry analysis.) The wash buffer can also be used to rapidly neutralize the alkaline pH of the lysis solution. any wash fluid or buffer is included in the kit, for example, the wash buffer could be distilled water In another embodiment, the wash buffer could be a pH buffer capable of maintaining a suitable pH for microorganisms, such as a phosphate buffer, MOPS or TRIS. For example, the wash buffer could be a phosphate solution of 0.01 M to about 0.2 M, pH 6.0 to 7.5

In addition, the kit may also comprise a filtration device, a ford source and / or a ford interface. The filtration device can be adapted for attachment to a ford source or a ford system, which functions to provide a ford for filtration (i.e. for ford filtration). In general, any means known in the art can be used to connect the sample filtration and transfer device to the ford system. For example, the filtration and transfer device can be connected to a ford system with the use of a simple ford tube, as is known in the art. For example, the kit may comprise a vial to the side arm ford (see Figures 2-3, number 2) with reusable filter holder or a connector (see Figure 3, number 4) with a filter reservoir and a plurality of holder -Reusable filters (see Figure 3, number 6). In still other embodiments, the kit may also comprise additional components for filtration, for example, the kit may comprise one or more tubes, clamps, valves, or the kit may comprise one or more tubes, clamps, valves or ford traps. In another embodiment, the kit may comprise one or more disposable filtration devices that have a built-in filtration membrane. In these disposable devices, filtration can be activated either by a ford source or by centrifugation.

The kit may also include a container (eg, a tube), within which the lysis step can be carried out. The container can be any container with sufficient volume to contain a test sample and optionally a lysis solution. In one embodiment, the container may be a tube. In another embodiment, the kit may include the separation device described in the related US patent application. No. 12 / 589,969 (currently published as US 2010/0120133 Al), filed on October 30, 2009, with the title "Separation Device for Use in the Separation, Characterization and / or Identification of Microorganisms." be from about 0.1 ml to about 25 ml, e.g., from about 1 ml to about 10 ml, e.g., from about 2 ml to about 8 ml. If the lysis stage and the subsequent isolation stage or separation are performed at a microscale, the volume of the container can be from about 2 pl to about 100 pl, eg, from about 5 pl to about 50 pl. The container can have a fixed closing device or can have a thread for accepting a closure device (e.g., a lid) so that the container may be tightly sealed during use The presence of a closure reduces the risks of handling microorganisms that are or may be infectious and / or dangerous rosos, as well as the risk of contaminating the sample. For example, the kit may contain from 1 to 500 containers (eg tubes) within which the lysis step can be carried out. In other embodiments, the kit can contain from 1 to 100, from 10 to 80, or from 10 to 50 containers within which the lysis step can be carried out. In other embodiments, the kit may contain 20, 50, 75 or 100 containers within which the lysis step can be performed.

The kit may also include one or more test slides or target plates for the analysis or examination of the microorganisms (eg, by mass spectrometry). According to this embodiment, the microorganisms accumulated in the integrated filtration and transfer device can be applied or directly deposited on or on a test slide or target plate for subsequent tests. According to this embodiment, the microorganisms isolated, accumulated and / or purified in the integrated filtration and transfer device can be transferred or deposited on a slide or a plate (e.g., using a small quantity application technique, reverse flow and / or extension) on the slide or target plate to allow further experimentation and / or analysis, as described in more detail here. For example, the kit may contain 1 to 500 test slides or target plates. In other embodiments, the kit may contain 1 to 100, or 10 to 80, or 20 to 50 test slides or target plates. In other embodiments, the kit may contain 20, 50, 75 or 100 test slides or target plates for further experimentation and / or analysis.

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In general, the kit can be configured to process any number of test samples (ie, for isolation, accumulation and / or purification of microorganisms from any specific number of test samples). For example, the kit can be configured to process (isolate, accumulate and / or purify microorganisms) from about 1 to about 1000 test samples, from about 10 to about 500 test samples, or from about 10 to about 100 test samples. In another example, the kit could be configured to process from about 10 to about 80 test samples, from about 20 to about 60 test samples, or about 50 test samples.

The present invention is further detailed in the following examples, which are offered by way of illustration and are not intended to limit the invention in any way. Conventional and widely known techniques that are specifically described below are used.

Examples

Example 1. Lysis-filtration and mass spectrometry through the use of an integrated filtration and transfer device

Microorganisms with an inoculum of approximately 40 or 400 CFU were "seeded" in a 1.0 ml suspension of each test strain in BacT / ALERT® SA or SN bottles containing 10 ml of human blood. The seeded test samples were mixed after turning the bottles several times, they were incubated in a BacT / ALERT® 3D Combo booth and monitored for microbial growth inside the bottle.

Blood culture broth samples were removed from the bottles a few minutes after being marked as positive by the BacT / ALERT® 3D Microbial Detection System. If the bottle was not processed immediately, it was stored at 2-8 ° C, and later it was brought to room temperature by placing the bottle in a 37 ° C water bath for 5-15 minutes before the test. Positive blood culture broth samples were processed to separate the microorganisms from the blood components and the medium that could interfere with the subsequent analysis, as follows:

(1) using a 1 ml syringe and 18G needle, 0.5 mL of positive broth was transferred to a clean 1.5 ml microfuge tube;

(2) 0.25 ml of lysis buffer (0.45% weight / volume Brij-97 + 0.3M CAPS, pH 11.7 (filtered through 0.2 µm filter) was added to the broth , and stored at 2-8 ° C)) and mixed by gentle aspiration / supply 5-6 times, carefully to avoid the formation of bubbles as much as possible, and the mixture was incubated for 2:00 to 2:15 minutes at room temperature, whereby a lysate sample or a lysate was generated;

(3) After incubation and lysis, the tip of an integrated filtration and transfer device was immersed approximately 3-5 mm in the lysate and aspirated to the ford for 2:00 to 2:15 minutes at room temperature, at as the liquid was carried to the bottom of the integrated filtration and transfer device, and adjusted to maintain a depth of approximately 3-5 mm;

(4) After the sample was filtered in a vacuum for 2:00 - 2:15 minutes, the filtration and transfer device was transferred to a container containing a first wash solution (Brij / Salina (0.45% weight / volume of NaCl + 0.05% of Brij 97)), was submerged, and aspirated to the ford for 4:00 - 4:15 minutes at room temperature;

(5) After the first washing step, the filtration and transfer device was transferred to a second container containing a second washing solution (deionized water), immersed in the first washing solution, and aspirated to the vat during 4:00 - 4:15 minutes at room temperature;

(6) The washed microbes were then applied to one or more staining points on the MALDI-TOF target plates using a technique of vertical application of small portions repeatedly, or alternatively by flow inversion, until a visible residue was deposited 1-2 pl of suspension;

(7) the points were dried and then 1pl of matrix was added;

(8) After applying and drying all samples for a given target plate, the spotting points were analyzed by mAlDI-TOF MS.

Example 2: Lysis-filtration and mass spectrometry through the use of an integrated filtration and transfer device comprising membrane filters

Forty-four (44) isolates of microorganisms were developed, processed and analyzed by MALDI-TOF MS, as described in Example 1, using an integrated sample filtration and transfer device using Supor® 450 (Pall-Gelman, Port Washington, NY) as a filtering material, and a flow inversion technique to deposit the accumulated microorganism on the MALDI-TOF plate.

Once all the microorganism samples had dried completely, mass spectra were obtained by MALDI-TOF for each in a mass / load range of 2,000-34,000 in a MALDI-TOF Axima Assurance mass spectrometer (Shimadzu Biotech North America, Maryland).

After obtaining each mass spectrum, a table of mass peaks was introduced in the "Sammis" microorganism identification software (bioMerieux Inc., USA) for analysis. This software consists of a database of MALDI-TOF mass spectra collected from microorganisms developed in agar.

Table 1 below shows the results of the identification (ID) through the use of a filtering and transfer device integrated with a Supor 450 membrane.

10 Table 1 - Supor 450 Device - Flow Investment Technique - Frascos SA

 Insulated material

 ID trust Isolated material ID trust

 E. coli, 104471

 3 S. epidermidis, D055 2

 E. coli, 100257

 4 S. epidermidis, D069 0

 E. coli, D104

 4 S. epidermidis, D082 4

 E. coli, D168

 4 S. epidermidis, D092 3

 P. aeroginosa, 105716

 4 E. faecalis, D149 4

 P. aeroginosa, 107930

 4 E. faecalis, D135 3

 E. aerogenes, 104098

 3 E.faecium, 13243 0

 E. aerogenes, 107930

 3 E.faecium, 14334 0

 K. pneumoniae, 108902

 4 E. faecium, D068 0

 K. pneumoniae, 105245

 4 S.pneumoniae, 7265 2

 S. aereus, 10754

 0 S.pneumoniae, 14226 0

 S. aereus, 8816

 4 S.pneumoniae, D013 0

 S. aereus, 7537

 4 S. pyogenes, 11629 0

 S. aereus, 7623

 0 S. pyogenes, 11620 0

 S. aereus, D164

 2 S. pyogenes, 11631 0

 S. aereus, D076

 2 S. pyogenes, 12897 0

 S. aereus, D116

 3 C. albicans, 303070 4

 S. aereus, D176

 2 C. albicans, 18804 3

 S. epidermidis, 13298

 0 C. albicans, 302611 4

 S. epidermidis, D011

 0 C. albicans, 304765 0

 S. epidermidis, D036

 3 C. albicans, 304771 0

 S. epidermidis, D042

 0 C. albicans, 304776 3

ID 4 confidence scores = 99.9%

3 = 85-99.8%

15 2 = Species ID <85%, or Gender or Family> 85%

0 = No ID

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As shown in Table 1, 22/44 (50%) received a correct ID with good confidence, while 61% identified at least some level.

Although the success rate of this experiment was not yet perfect, it shows that the concept is very promising. This set of microorganisms was chosen because they challenged the direct identification from the blood culture using previous centrifugal methods and, therefore, provided a rigorous test.

There are many reasons why these first trials may not have provided correct IDs in all cases. One possibility is that there was not enough cell mass, another possibility is that the deposited cells had insufficient purity, or a combination of both circumstances could have occurred.

If the problem is cell mass, then other membranes, or even deep filters still not investigated, can capture a larger number of cells. Another possibility is to increase the ratio time / volume of capture to accumulate a larger mass.

Example 3: Lysis-filtration mass spectromethane using an integrated filtration and transfer device comprising depth filters.

Forty-six (46) microorganism isolates were developed, processed and analyzed by MALDI-TOF MS as described in Example 1, using an integrated sample filtration and transfer device that employs depth filters (Whatman GF / F filter ( 0.7 nominal micrometers) and Metrigard filters (Pall-Gelman) (0.5 nominal micrometers with acrylic binder)) as filtering material instead of membranes.

Integrated filtration and sample transfer devices made with the Pall-Gelman Metrigard filters (0.5 micrometer nominal with acrylic binder) were tested only on a few of the most problematic samples, but gave results as good as the GF filters / F or better.

Once all the microorganism samples had dried completely, MALDI-TOF mass spectra were obtained for each in a mass / load range of 2,000-34,000 in a MALDI-TOF Axima Assurance mass spectrometer (Shimadzu Biotech North America , Maryland).

After obtaining each mass spectrum, a table of mass peaks was introduced in the "Sammis" microorganism identification software (bioMerieux Inc. USA) for analysis. This software consists of a database of MALDI-TOF mass spectra collected from microorganisms developed in agar.

Table 2 below shows the ID results by using an integrated filtering and transfer device comprising a Whatman GF / F filter or Pall-Gelman Metrigard filters.

Table 2 - Crops planted in SA bottles processed with depth filter type device

 Insulated material

 GF / F device Insulated material GF / F device Metrigard device

 E. coli, 104471

 4 S. epidermidis, D055 3 ND

 E. coli, 100257

 4 S. epidermidis, D069 3 ND

 E. coli, D104

 4 S. epidermidis, D082 4 ND

 E. coli, D168

 4 S. epidermidis, D092 4 ND

 P. aeroginosa, 105716

 4 E. faecalis, D149 4 ND

 P. aeroginosa, 107930

 4 E. faecalis, D135 4 ND

 E. aerogenes, 104098

 4 E.faecium, 13243 4 ND

 E. aerogenes, 107930

 4 E.faecium, 14334 4 ND

 K. pneumoniae, 108902

 4 E. faecium, D068 3 ND

 K. pneumoniae, 105245

 4 S.pneumoniae, 7265 3 ND

 S. aereus, 10754

 3 S.pneumoniae, 41226 3 3

 S. aereus, 8816

 4 S.pneumoniae, D0013 3 ND

 S. aereus, 7537

 4 S.pneumoniae, D002 3 ND

5

10

fifteen

twenty

25

30

35

 S. aereus, 7623

 4 S. pyogenes, 11629 2 3

 S. aereus, D164

 4 S. pyogenes, 11620 0 2

 S. aereus, D076

 4 S. pyogenes, 11631 3 ND

 S. aereus, D116

 4 S. pyogenes, 12897 3 ND

 S. aereus, D176

 4 C. albicans, 303070 4 ND

 S. epidermidis, 13298

 3 C. albicans, 18804 4 ND

 S. epidermidis, D011

 4 C. albicans, 302611 2 4

 S. epidermidis, D036

 3 C. albicans, 304765 4 ND

 S. epidermidis, D042

 4 C. albicans, 304771 3 ND

 C. albicans, 304776 4 ND

ID 4 confidence scores = 99.9%

3 = 85-99.8%

2 = Species ID <85%, or Generate or Family> 85%

0 = No ID

ND = Not determined

Table 2 shows the results of sown crops that were developed in SA bottles and processed with a Filter Device made from depth filters instead of membranes. In general, the results are very good and similar to the best that have been obtained by other processing methods. The device made with Whatman GF / F filter (0.7 nominal micrometers) gave a confidence ID (> 85% confidence) with respect to the level of species (including the typical results of low discrimination with S.pneumoniae and C. albicans) with 43 of 46 isolates (93.5%), and ID for at least gender or species at a lower confidence level, with 45 of 46 isolates (97.8%).

Therefore, the use of depth filters shows an improvement over previous experiments with membrane filters. It is possible that the greater amount of cells that can be trapped in a depth filter contribute to better results, but another factor could also be the improvement in washing. Gram and / or cell strain counts of captured organisms can help distinguish between the two possibilities.

Another difference with respect to previous trials is that the MALDI-TOF acquisition program was readjusted (as is necessary periodically due to the aging of the instrument / detector) between the two experiments. The readjustment would probably have also improved the results of the membrane device, but does not exclusively represent the magnitude of the difference observed with the depth filters.

Example 4: Lysis-filtration and mass spectrometry through the use of an integrated filtration and transfer device comprising depth filters with medium bottles containing resin.

Fifty-one (51) isolates of microorganisms were developed, processed and analyzed by MALDI-TOF MS as described in Example 3, with the use of an integrated sample filtration and transfer device employing Metrigard® depth filters except that a) the crops planted were grown in bottles containing aerobic media with resins instead of the BacT / ALERT® SA bottles, b) the lysis buffer had the following composition (0.6% by weight / volume of Brij-97 + 0.4M in CAPS, pH 11.7), and c) the first wash solution has the following composition (20 mM sodium phosphate, 0.05% by weight / volume of Brij 97, 0.45% by weight / NaCl volume, pH 7.2).

Once all the microorganism samples had dried completely, mass spectra were obtained by MALDI-TOF for each in a mass / load range of 2,000-34,000 in a MALDI-TOF Axima Assurance mass spectrometer (Shimadzu Biotech North America, Maryland).

After obtaining each mass spectrum, a table of mass peaks was introduced in the "Sammis" microorganism identification software (bioMerieux Inc. USA) for analysis. This software consists of a database of MALDI-TOF mass spectra collected among microorganisms developed in agar.

Table 3 below shows the results of ID through the use of a filtering and transfer device 5 integrated with Pall-Gelman Metrigard filters from cultures developed in aerobic media containing resin.

Table 3 - Crops sown in bottles with aerobic resin media processed with depth filter type device

 Insulated material

 ID trust Isolated material ID trust

 E. coli, 104471

 4 S. epidermidis, 13298 4

 E. coli, 100257

 4 S. epidermidis, D011 4

 E. coli, D104

 4 S. epidermidis, D036 4

 E. coli, D168

 4 S. epidermidis, D055 4

 E. coli-101262

 4 S. epidermidis, D069 4

 E. aerogenes, 104098

 4 S. epidermidis, D082 4

 E. aerogenes, 107930

 4 S. epidermidis, D092 4

 K. pneumoniae, 108902

 4 E. faecalis, D149 4

 K. pneumoniae, 105245

 4 E. faecalis, D135 4

 K. pneumoniae -104143

 3 E. faecalis -15268 4

 K. pneumoniae -108904

 3 E. faecalis - 15282 4

 P. aeruginosa, 105716

 4 E.faecium, 13243 4

 P. aeruginosa, 108042

 4 E.faecium, 14334 4

 P. aeruginosa -104014

 4 E. faecium, D050 4

 P. aeruginosa-108937

 4 E.faecium, D068 4

 S. aereus, 10754

 4 S.pneumoniae, 7265 3

 S. aereus, 8816

 4 S.pneumoniae, 14226 3

 S. aereus, 7537

 4 S.pneumoniae, D013 3

 S. aereus, 7623

 4 S.pneumoniae, D002 3

 S. aereus, D164

 4 S. pyogenes, 11629 3

 S. aereus, D076

 4 S. pyogenes, 11620 3

 S. aereus, D116

 4 S. pyogenes, 11631 3

 S. aereus, D176

 4 S. pyogenes, 12897 1

 S. albicans, 303070

 4 H. influenzae, 500891 3

 S. albicans, 304771

 2 H. influenzae, 500893 0

 S. albicans, 304776

 0

ID 4 confidence scores = 99.9%

3 = 85-99.8%

2 = species ID <85%, or generates or family> 85%

5 0 = No ID

As shown in Table 3, the results of sown crops that were developed in bottles containing resin and that were processed with Filter Device with depth filters are very good. These processed crops gave a confidence ID (> 85% confidence) with respect to the species level (including typical results of low discrimination with S.pneumoniae and C. albicans) with 47 of 51 isolates (92.2%).

10

Claims (14)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 1. An integrated sample filtration and transfer device comprising: an elongated and hollow body having a first end or tip that is provided with, or covered with, a filtration material, wherein said filtration material is located adjacent to and protrudes from said first end or tip; wherein said elongated and hollow body is filled or plugged with an adsorbent to provide support for the filtration material; Y a second operable end to provide fluid flow for filtration. 2. The integrated sample filtration and transfer device of claim 1, wherein said second end is adapted for connection to a ford system, a syringe or a plunger to generate a ford. 3. The integrated sample filtration and transfer device of claims 1 or 2, wherein said elongated and hollow body is made of a plastic material nidd or semi-nigid selected from the group of polypropylene (PP), polycarbonate (PC), polyethylene terephthalate (PET), or other plastic material. 4. The integrated sample filtration and transfer device of claims 1 to 3, wherein said elongated and hollow body comprises an elongated and cylindrical body that has a diameter of 0.5 mm to 10 mm and a length of 2 cm to 20 cm. 5. The integrated sample filtration and transfer device of claims 1 to 4, wherein said elongated and hollow body has an internal volume of 0.5 cm3 to 10 cm3. 6. The integrated sample filtration and transfer device of claims 1 to 5, wherein said adsorbent provides passive filtration by a capillary action or absorption force for fluid flow. 7. The integrated sample filtration and transfer device of claim 6, wherein said adsorbent is selected from the group consisting of cotton, polyester, an adsorbent resin, a silica gel, a hydrogel, a molecular sieve, zeolite, or other adsorbent materials. 8. The integrated sample filtration and transfer device of claims 1 to 7, wherein said filtration material has a pore size of 0.1 pm to 10.0 pm. 9. The integrated sample filtration and transfer device of claims 1 to 8, wherein said filtration material is selected from the group consisting of polyethersulfone (PES) membranes, polysulfone, mixed cellulose ester, PVDF, polycarbonate, materials deep filters selected from the group consisting of fiberglass types GF / F, GF / C and GMF150, fiberglass, AP15 glass fiber, as well as a variety of cellulose, polyester, polypropylene or other fiber filters and particulate material, or a combination thereof 10. A sample filtration and transfer unit comprising: a plurality of integrated sample filtration and transfer devices of claims 1 to 9, for the isolation and / or accumulation of a plurality of test samples and for transfer simultaneous with the plurality of microorganisms isolated and / or accumulated in a container, slides or plate to analyze said microorganisms isolated or accumulated. 11. The sample filtration and transfer unit of claim 10, wherein said unit further comprises: a base plate and a separate upper plate of said base plate, wherein optionally at least one support rod or a pair of vertical support rods located between said upper plate and said base plate and separating them is provided; a rack having a plurality of wells to contain a plurality of individual tubes, wherein said rack is optionally supported through a pair of base rails and a corresponding pair of rod rods of the rack; a vertical axis arm, optionally associated with a vertical platform, operable to allow said vertical axis arm to move "up" and "down", optionally along the vertical platform, to raise and lower said upper plate and said plurality of integrated filtration and sample transfer devices; Y a forging unit comprising an alignment bar containing a plurality of holes or cavities for containing a plurality of integrated and removable sample filtration and transfer devices according to claims 1 to 9, and a valve manifold associated with a plurality of valves and connectors where each individually holds and connects to said integrated filtration and sample transfer devices. 5 10 fifteen twenty 25 12. A method for isolating and identifying a microorganism from a test sample, comprising: (a) provide a test sample that is known to contain or may contain microorganisms; (b) optionally lysing cells and / or non-microbial particulate material in said test sample to produce a lysed sample; (c) isolating and accumulating said microorganisms from other components of said test sample or said sample lysed by filtration, using the integrated sample filtration and transfer device of claims 1-9; (d) transferring said sample of isolated microorganisms to a suitable container or slide to analyze and / or examine said isolated and accumulated sample of microorganisms; (e) analyze said isolated or accumulated sample of said microorganisms to obtain measurements for the characterization and / or identification of the microorganisms; Y (f) characterize and / or identify said microorganisms in said isolated and accumulated sample based on the acquired measurements. 13. The method of claim 12, wherein said analysis step (e) is by mass spectrometry and wherein said spectromethane is selected from the group consisting of MALDI-TOF mass spectrometry, mass spectrometry with electrospray ionization and desorption (DESI), mass spectrometry coupled to gas phase chromatography, mass spectrometry coupled to liquid phase chromatography, mass spectrometry with electrospray ionization (ESI), and spectrometry with selected ion flow tube (SIFT). 14. The method of claims 12 to 13, wherein said sample is a thermal or non-thermal sample and wherein, when said sample is a thermal sample, then the thermal sample is selected from the group consisting of blood, serum, plasma , bloody fractions, joint fluid, urine, semen, saliva, feces, cerebrospinal fluid, gastric contents, vaginal secretions, tissue homogenates, bone marrow aspirates, bone homogenates, sputum, aspirates, swabs and rinses of swabs, body fluids, a sample of blood culture, and a urine sample.